FIG 7.

HCV induces polyubiquitylation of VPS4A via the K6-, K11-, K27-, and K29-linkage polyubiquitin chains. (A and B) Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. At 3 h postinfection, cells were cotransfected with pcDNA-VPS4A-FLAG and the indicated HA-tagged Ub plasmids. At 4 days transfection, cells were harvested, and cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (1st panel) or anti-FLAG PAb (2nd panel). Input samples were immunoblotted with anti-HA PAb (3rd panel), anti-FLAG PAb (4th panel), anti-NS3 MAb (5th panel), or anti-GAPDH MAb (6th panel), respectively. The level of GAPDH served as a loading control. HC, immunoglobulin heavy chain. (C) HCV-infected cells and mock-infected control cells were transfected with pcDNA-VPS4A-FLAG. The cells were treated with 50 μg/mL cycloheximide (CHX) at 48 h after transfection. The cell lysates were harvested at 0, 8, 16, and 24 h after treatment with CHX, followed by immunoblotting with anti-FLAG PAb (top), anti-p53 MAb (middle), and anti-GAPDH MAb (bottom). The level of GAPDH served as a loading control. (D) Specific signals were quantitated by densitometry, and the percentage of remaining VPS4A-FLAG, p53, and GAPDH at each time was compared with that at the starting point, respectively. Closed triangles, HCV-infected cells; closed circles, mock-infected control cells. Red lines, blue lines, and black lines indicate VPS4A-FLAG, p53, and GAPDH, respectively. H, HCV; M, mock.